Dual selective PI3 delta and gamma kinase inhibitors

ABSTRACT

The present invention relates to dual delta (δ) and gamma (γ) PI3K protein kinase modulators, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prevention and/or amelioration of Pi3K kinase mediated diseases or disorders with them.

The present application claims the benefit of Indian Patent ApplicationNos. 2501/CHE/2013, filed Jun. 7, 2013, and 5567/CHE/2013, filed Dec. 3,2013 each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides dual delta (δ) and gamma (γ) PI3K proteinkinase modulators, methods of preparing them, pharmaceuticalcompositions containing them and methods of treatment, prevention and/oramelioration of Pi3K kinase mediated diseases or disorders with them.

BACKGROUND OF THE INVENTION

Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellularlipid kinases that phosphorylate the 3 position hydroxyl group of theinositol ring of phosphoinositide lipids (PIs) generating lipid secondmessengers. While alpha and beta isoforms are ubiquitous in theirdistribution, expression of delta and gamma is restricted to circulatinghematogenous cells and endothelial cells. Unlike PI3K-alpha or beta,mice lacking expression of gamma or delta do not show any adversephenotype indicating that targeting of these specific isoforms would notresult in overt toxicity.

Recently, targeted inhibitors of the phosphoinositide-3-kinase (PI3K)pathway have been suggested as immunomodulatory agents. This intereststems from the fact that the PI3K pathway serves multiple functions inimmune cell signaling, primarily through the generation ofphosphatidylinositol (3,4,5)-trisphosphate (PIP3), a membraneboundsecond messenger. PIP3 recruits proteins to the cytoplasmic side of thelipid bilayer, including protein kinases and GTPases, initiating acomplex network of downstream signaling cascades important in theregulation of immune cell adhesion, migration, and cell-cellcommunication.

The four class I PI3K isoforms differ significantly in their tissuedistribution. PI3Kα and PI3Kβ are ubiquitous and activated downstream ofreceptor tyrosine kinases (RTK), whereas PI3K δ and PI3K γ are primarilylimited to hematopoietic and endothelial cells, and are activateddownstream of RTKs, and G protein coupled receptors (GPCR),respectively. Mouse genetic studies have revealed that PI3Kα and PI3Kβare essential for normal development, whereas loss of PI3K δ and/or PI3Kγ yields viable offspring with selective immune deficits.

The expression pattern and functions of PI3K δ and PI3K γ have generatedmuch interest in developing PI3Kδ/γ inhibitors as agents for manydiseases, including rheumatoid arthritis, allergies, asthma, chronicobstructive pulmonary disease and multiple sclerosis (Hirsch et al.,Pharmacol. Ther., 118, 192-205, 2008; Marone et al., Biochim. Biophys.Acta., 1784, 159-185, 2008; Rommel et al., Nat. Rev. Immunol., 7,191-201, 2007; Ruckle et al., Nat. Rev. Drug Discov., 5, 903-918, 2006).Studies using both pharmacologic and genetic methods have shown thesetwo isoforms often demonstrate synergistic interactions with each other(Konrad et al., J. Biol. Chem., 283, 33296-33303, 2008; Laffargue etal., Immunity, 16, 441-451, 2002). In mast cells, for example, PI3Kδ isessential for degranulation in response to IgE cross-linking ofFc-receptors (Ali et al., J. Immunol., 180, 2538-2544, 2008), but PI3Kγplays an important role in amplifying the response (Laffargue et al.,Immunity, 16, 441-451, 2002). Similar effects have been seen in othercellular functions, including lymphocyte homing and the neutrophilrespiratory burst where PI3Kγ plays a critical role and PI3Kδ amplifieseach process. The nonredundant but related roles of PI3Kδ and PI3Kγ havemade it difficult to determine which of the two isoforms (alone or incombination) is best targeted in a particular inflammatory disorder.Studies using mice that lack PI3Kδ and/or PI3Kγ or express kinase-deadvariants of PI3Kδ and PI3Kγ have been valuable tools in understandingtheir roles. For example, PI3Kδ knockout mice demonstrated diminishedneutrophil chemotaxis, diminished antibody production (both T celldependent and independent) (Jou et al., Mol. Cell. Biol., 22, 8580-8591,2002), and lower numbers of mature B cells (Clayton et al., J. Exp.Med., 196, 753-763, 2002; Jou et al., Mol. Cell. Biol., 22, 8580-8591,2002), and a decrease in their proliferation in response to anti-IgM(Jou et al., 2002). This phenotype was replicated in the PI3Kδkinase-dead variant and with PI3Kδ selective inhibitors along withdecreased numbers of and proliferation of mast cells, and an attenuatedallergic response. The PI3Kγ knockout contained higher numbers of, butless responsive, neutrophils, lower numbers of and less responsivemacrophages and dendritic cells displayed decreased mast celldegranulation (Laffargue et al., 2002), a higher ratio of CD4+ to CD8+ Tcells), increased thymocyte apoptosis, diminished induction of CXCR3 onactivated T cells and decreased cardiac contractility. This lattereffect on cardiac tissue was a concern for chronic dosing of patientswith PI3Kγ inhibitors. However, this concern was largely mitigated whenthe PI3Kγ kinase-dead variant (which better mimics inhibition of thekinase rather than loss of the protein) showed similar immune cellphenotypes, but importantly had no cardiac defects. The cardiac effectwas later shown to be due to scaffolding effects rather than thecatalytic activity of PI3Kγ. The dual PI3Kδ/PI3Kγ knockout was viablebut exhibited serious defects in T cell development and thymocytesurvival. The PI3Kγ knockout/PI3Kδ kinase-dead combination produced asimilar phenotype suggesting that at least within the immune system, therole of PI3Kδ is likely only a catalytic one. Interpretation of studiesusing knockout and kinase-dead mice can be challenging because thesemodels provide only a steady-state picture of the immune system, lacktemporal and dose control, and do not permit a full understanding of howa dynamic immune response will react to reversible inhibition. Selectiveinhibitors with varying profiles (PI3Kδ, PI3Kγ, and PI3Kδ/γ) arenecessary for studies of leukocyte signaling in order to assess therelative contributions of each PI3K to immune cell activation (Olusegonet al., Chemistry & Biology, 1, 123-134 (2010), including the referencescited therein)

Dual inhibition of δ/γ is strongly implicated as an interventionstrategy in allergic and non-allergic inflammation of the airways andother autoimmune diseases. Scientific evidence for PI3K-δ and γ gammainvolvement in various cellular processes underlying asthma and COPDstems from inhibitor studies and gene-targeting approaches. Also,resistance to conventional therapies such as corticosteroids in severalCOPD patients has been attributed to an up-regulation of the PI3K δ/γpathway. Disruption of PI3K-δ/γ signalling therefore provides a novelstrategy aimed at counteracting the immuno-inflammatory response. Due tothe pivotal role played by PI3K-δ and γ in mediating inflammatory cellfunctionality such as leukocyte migration and activation, and mast celldegranulation, blocking these isoforms may also be an effective strategyfor the treatment of rheumatoid arthritis as well. Given the establishedcriticality of these isoforms in immune surveillance, inhibitorsspecifically targeting the δ and γ isoforms would be expected toattenuate the progression of immune response encountered in airwayinflammation and rheumatoid arthritis (William et. al Chemistry &Biology, 17, 123-134, 2010 and Thompson, et al. Chemistry & Biology,17:101-102, 2010)

Reviews and studies regarding PI3K and related protein kinase pathwayshave been given by Liu et. al., Nature Reviews Drug Discovery, 8,627-644, 2009); Nathan T. et. al., Mol Cancer Ther., 8(1), 2009; Maroneet, al., Biochimica et Biophysica Acta, 1784, 159-185, 2008 and Markmanet. al., Annals of Oncology Advance Access, published August 2009.Similarly reviews and studies regarding role of PI3K δ and γ have beengiven by William et. al., Chemistry & Biology, 17, 123-134, 2010 andTimothy et. al. J. Med. Chem., 55 (20), 8559-8581, 2012. All of theseliterature disclosures are hereby incorporated by reference in theirentirety.

Compounds such as IPI-145 and CAL130 have been reported as dualinhibitors of Pi3K δ/γ. IPI-145 is under clinical investigation forcancer, asthma and rheumatoid arthritis. IPI-45 have been reported tohave a maximum tolerated dose (MTD) of 75 mg BID (55th ASH® AnnulaMeeting New Orleans-LA, Dec. 7-10, 2013). There are no reports ofCAL-130 being investigated for clinical purposes.

There still remains an unmet need for dual δ γ PI3K modulators for thetreatment of diseases and disorders associated with δ/γ PI3Kkinases-mediated events.

Further reference is made herein to International Publication Nos. WO11/055,215 and WO 12/151,525 and U.S. Publication Nos. 2011/0118257 and2012/0289496, each of which is incorporated herein by reference in itsentirety.

SUMMARY OF THE INVENTION

The present invention is directed to selective dual inhibitors of PI3Kdelta and gamma protein kinases. These compounds are suitable for use ina pharmaceutical composition for the treatment of a PI3K associateddisease, disorder or condition, e.g., a proliferative disease such ascancer Inhibition of both PI3K delta and gamma protein kinases mayprovide beneficial effects in the treatment of certain diseases anddisorders.

The selective dual inhibitors of the present invention include thefollowing compounds, pharmaceutically acceptable salts thereof, andprodrugs thereof:

-   (RS)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one.    (Compound A)-   (S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one.    (Compound A1)-   (R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one.    (Compound A2)

The chemical structures of the compounds of the present invention areshown below.

In one embodiment, the present invention relates to the compound(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one,or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one,or a pharmaceutically acceptable salt thereof, is substantially free(e.g., contains less than about 10%, such as less than about 5%, lessthan about 2.5%, less than about 1%, less than about 0.1% by weight oris free) of(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneand pharmaceutically acceptable salts thereof.

In another embodiment, the compound(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one,or a pharmaceutically acceptable salt thereof, has an enantiomericexcess of greater than about 90%, such as greater than about 91%,greater than about 92%, greater than about 93%, greater than about 94%,greater than about 95%, greater than about 96%, greater than about 97%,greater than about 98%, greater than about 99%, greater than about99.5%, greater than about 99.9%, or greater than about 99.99%.

In one preferred embodiment, the present invention relates to thecompound(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one(Compound A1).

The invention further provides a pharmaceutical composition comprisingone or more compounds of the present invention (such as compound A1)together with a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may further comprise one or more of additional active agents(such as anti-cancer agents and the active agents discussed below). Inone embodiment, the pharmaceutical composition includes atherapeutically effective amount of one or more compounds of the presentinvention.

Another embodiment is a method of inhibiting PI3K delta and gamma in apatient by administering to the patient an effective amount of at leastone compound of the present invention.

Yet another embodiment is a method of treating, preventing, and/orinhibiting a PI3K protein kinase mediated disease, disorder or condition(such as cancer or other proliferative disease or disorder) in a patientby administering to the patient an effective amount of at least onecompound of the present invention.

Yet another embodiment of the present invention is a method forinhibiting PI3K, in particular PI3K delta and gamma kinase in a patientby administering to the patient an effective amount of at least onecompound of the present invention.

Yet another embodiment of the present invention is a method for treatingan inflammatory, autoimmune or proliferative disease via modulation of aprotein kinase (such as PI3 delta and gamma kinase) by administering toa patient in need of such treatment an effective amount of at least onecompound of the present invention. In one embodiment, the compound ofthe present invention inhibits both the PI3K delta and gamma proteinkinase.

Yet another embodiment of the present invention is a method for treatingan inflammatory, autoimmune or proliferative disease via modulation of aprotein kinase (such as PI3 delta and gamma kinase) by administering toa patient in need of such treatment an effective amount of at least onecompound of the present invention, in combination (simultaneously orsequentially) with at least one other anti-inflammatory, immunomodulatoror anti-cancer agent (or a combination thereof). In one embodiment, thecompound of the present invention inhibits both the PI3K delta and gammaprotein kinase.

The compounds of the present invention are useful in the treatment of avariety of cancers, including, but not limited to:

carcinoma, including, but not limited to, that of the bladder, breast,colon, kidney, liver, lung, including small cell lung cancer, esophagus,gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, andskin, including squamous cell carcinoma;

hematopoietic tumors of lymphoid lineage, including, but not limited to,leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkinslymphoma, hairy cell lymphoma and Burkett's lymphoma;

hematopoietic tumors of myeloid lineage, including, but not limited to,acute and chronic myelogenous leukemias, myelodysplastic syndrome andpromyelocytic leukemia;

tumors of mesenchymal origin, including, but not limited to,fibrosarcoma and rhabdomyosarcoma;

tumors of the central and peripheral nervous system, including, but notlimited to, astrocytoma, neuroblastoma, glioma and schwannomas; and

other tumors, including, but not limited to, melanoma, seminoma,teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma,thyroid follicular cancer and Kaposi's sarcoma.

In one embodiment, the compounds of the present invention areadministered to treat a leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burkett'slymphoma, acute and chronic myelogenous leukemias, myelodysplasticsyndrome and promyelocytic leukemia.

Due to the key role of protein kinases in the regulation of cellularproliferation in general, the protein kinase inhibitors of the presentinvention could act as reversible cytostatic agents, and may be usefultherefore in the treatment of any disease process which featuresabnormal cellular proliferation, e.g., benign prostatic hyperplasia,familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis,pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosisfollowing angioplasty or vascular surgery, hypertrophic scar formation,inflammatory bowel disease, transplantation rejection, endotoxic shock,and fungal infections.

The compounds of the present invention as modulators of apoptosis areuseful in the treatment of cancer (including but not limited to thosetypes mentioned herein above), viral infections (including, but notlimited to, herpevirus, poxvirus, Epstein-Barr virus, Sindbis virus andadenovirus), autoimmune diseases (including, but not limited, tosystemic lupus, erythematosus, autoimmune mediated glomerulonephritis,rheumatoid arthritis, psoriasis, inflammatory bowel disease, andautoimmune diabetes mellitus), neurodegenerative disorders (including,but not limited to, Alzheimer's disease, AIDS-related dementia,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration),myelodysplastic syndromes, aplastic anemia, ischemic injury associatedwith myocardial infarctions, stroke and reperfusion injury, arrhythmia,atherosclerosis, toxin-induced or alcohol related liver diseases,hematological diseases (including, but not limited to, chronic anemiaand aplastic anemia), degenerative diseases of the musculoskeletalsystem (including, but not limited to, osteoporosis and arthritis)aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis,kidney diseases and cancer pain. The compounds of the present inventionare also useful in the prevention, inhibition, or suppression of AIDSdevelopment in HIV-infected individuals.

The compounds of the present invention can modulate the level ofcellular RNA and DNA synthesis. These agents are therefore useful in thetreatment of viral infections, including, but not limited to, HIV, humanpapilloma virus, herpesvirus, poxvirus, Epstein-Barr virus, Sindbisvirus and adenovirus.

The compounds of the present invention are useful in the chemopreventionof cancer. Chemoprevention is defined as inhibiting the development ofinvasive cancer by either blocking the initiating mutagenic event or byblocking the progression of pre-malignant cells that have alreadysuffered an insult or inhibiting tumor relapse. The compounds of thepresent invention are also useful in inhibiting tumor angiogenesis andmetastasis. One embodiment of the invention is a method of inhibitingtumor angiogenesis or metastasis in a patient in need thereof byadministering an effective amount of one or more compounds of thepresent invention.

Another embodiment of the present invention is a method of treating animmune system-related disease (e.g., an autoimmune disease), a diseaseor disorder involving inflammation (e.g., asthma, chronic obstructivepulmonary disease (COPD), rheumatoid arthritis, inflammatory boweldisease, glomerulonephritis, neuroinflammatory diseases, multiplesclerosis, uveitis and disorders of the immune system), cancer or otherproliferative disease, a hepatic disease or disorder, a renal disease ordisorder. The method includes administering an effective amount of oneor more compounds of the present invention.

Examples of immune disorders include, but are not limited to, psoriasis,rheumatoid arthritis, vasculitis, inflammatory bowel disease,dermatitis, osteoarthritis, asthma, inflammatory muscle disease,allergic rhinitis, vaginitis, interstitial cystitis, scleroderma,osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ,bone marrow, stem cells and other cells and tissues) graft rejection,graft-versus-host disease, lupus erythematosus, inflammatory disease,type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren'ssyndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis),myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis,cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,allergic conjunctivitis and atopic dermatitis.

In one embodiment, the compounds described herein are useful asimmunosuppressants to prevent transplant graft rejections, allogeneic orxenogeneic transplantation rejection (organ, bone marrow, stem cells,other cells and tissues), and graft-versus-host disease. In otherembodiments, transplant graft rejections result from tissue or organtransplants. In further embodiments, graft-versus-host disease resultsfrom bone marrow or stem cell transplantation. One embodiment is amethod of preventing or decreasing the risk of transplant graftrejection, allogeneic or xenogeneic transplantation rejection (organ,bone marrow, stem cells, other cells and tissues), or graft-versus-hostdisease by administering an effective amount of one or more compounds ofthe present invention.

The compounds of the present invention are also useful in combination(administered together or sequentially) with known anti-cancertreatments, such as radiation therapy or with cytostatic or cytotoxic oranticancer agents, such as, for example, DNA interactive agents, such ascisplatin or doxorubicin; topoisomerase II inhibitors, such asetoposide; topoisomerase I inhibitors such as CPT-11 or topotecan;tubulin interacting agents, such as paclitaxel, docetaxel or theepothilones (for example ixabepilone), either naturally occurring orsynthetic; hormonal agents, such as tamoxifen; thymidilate synthaseinhibitors, such as 5-fluorouracil; and anti-metabolites, such asmethotrexate, other tyrosine kinase inhibitors such as Iressa andOSI-774; angiogenesis inhibitors; EGF inhibitors; VEGF inhibitors; CDKinhibitors; SRC inhibitors; c-Kit inhibitors; Her1/2 inhibitors andmonoclonal antibodies directed against growth factor receptors such aserbitux (EGF) and herceptin (Her2) and other protein kinase modulatorsas well.

The compounds of the present invention are also useful in combination(administered together or sequentially) with one or more steroidalanti-inflammatory drugs, non-steroidal anti-inflammatory drugs (NSAIDs)or immune selective anti-inflammatory derivatives (ImSAIDs).

The invention further provides a pharmaceutical composition comprisingone or more compounds of the present invention together with apharmaceutically acceptable carrier. The pharmaceutical composition mayfurther comprise one or more of the active ingredients identified above,such as other anti-cancer agents.

Yet another embodiment is a method of treating leukemia in a patient inneed thereof by administering a therapeutically effective amount of acompound of the present invention. For example, the compounds of thepresent invention are effective for treating chronic lymphocyticleukemia (CLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) acutemyeloid leukemia (AML), multiple myeloma (MM), small lymphocyticlymphoma (SLL), and indolent non-Hodgkin's lymphoma (I-NHL).

Yet another embodiment is a method of treating leukemia in a patient inneed thereof by administering a therapeutically effective amount of acompound of the present invention. For example, the compounds of thepresent invention are effective for treating autoimmune disorders suchas asthma, COPD, rhematoid arthritis, psorias, lupus and experimentalautoimmune encephalomyelitis (EAE).

Yet another embodiment is a method of treating allergic rhinitis in apatient in need thereof by administering a therapeutically effectiveamount of a compound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a bar graph of the neutrophil count in bronchioalveolarlavage fluid (BALF) from animals treated with 0, 0.3, 1, 3, and 10 mg/kgof Compound A1 (po) according to the Lipopolysaccharide inducedpulmonary neutrophilia model described in Assay 7.

FIG. 2 depicts a bar graph of the neutrophil count in peritoneal lavagefluid from animals treated with 0, 1, 3, and 10 mg/kg of Compound A1(po) according to the Lipopolysaccharide-induced rat air pouchinflammation model described in Assay 8.

FIG. 3 depicts a bar graph of the Lipopolysaccharide-induced plasmaTNF-α concentration in fasted female Wistar rats followingadministration of 0, 1, 3, and 10 mg/kg of Compound A1 (po) according tothe procedure described in Assay 9.

FIGS. 4A and 4B depict graphs of enhanced pause (Penh) or PEF/PIF (peakexpiratory flow/peak inspiratory flow) ratio, respectively, insensitized male guinea pigs following methacholine challenge andtreatment with OVA/SAL or OVA/OVA or 0.3, 1, or 3 mg/kg Compound A1according to the procedure in Assay 10A.

FIGS. 4C-4E depict bar graphs of eosinophil count in BALF, total cellcount in BALF, and percentage eosinophils, respectively, inovalbumin-sensitized male guinea pigs and treatment with 0, 0.3, 1, or 3mg/kg Compound A1 according to the procedure in Assay 10A.

FIGS. 5A and 5B depict graphs of Penh) or PEF/PIF ratio, respectively,in ovalbumin-sensitized mice following methacholine challenge andtreatment with SAL/SAL, OVA/SAL or OVA/OVA or 3 mg/kg Compound A1according to the procedure in Assay 10B.

FIGS. 5C-5E depict bar graphs of eosinophil count in BALF, total cellcount in BALF, and percentage eosinophils, respectively, inovalbumin-sensitized mice and treated with 0 or 3 mg/kg Compound A1according to the procedure in Assay 10B.

FIGS. 6A and 6B depict bar graphs of individual histopathological scoresfor ankle and knee, respectively, in collagen induced arthritis usingLewis rats treated with a control or 15 mg/kg/BID of compound A1according to the procedure in Assay 11.

FIGS. 6C and 6D depict bar graphs of summed histopathological scores forankle and knee, respectively, in collagen induced arthritis model usingLewis rats treated with vehicle or 15 mg/kg/BID of compound A1 accordingto the procedure in Assay 11.

FIGS. 7A and 7B depict bar graphs of macrophage and neutrophil cellcounts, respectively, in BALF following administration of 0.3, 1, or 3mg/kg/BID of Compound A1 in male Balb/c mice in a cigarette smokeinduced cell infiltration model as described in Assay 15.

FIG. 8 depicts a graph showing the inhibition of AKT phosphorylation inleukemic cell lines (MOLT-4, Jurkat, CCRF-CEM, Hut-78, and HuT-102) byCompound A1 according to the procedure in Assay 3.

FIG. 9 depicts a graph showing the inhibition in percentage of CD63positive cells induced by fMLP or anti-FcεR1 in human whole blood byCompound A1 according to the procedure in Assay 4.

FIG. 10 depicts a graph showing the inhibition of anti-humanCD3/CD28-induced cytokines (TNFα, IFNγ and IL2) by Compound A1 accordingto Assay 6D.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following definitions shall apply unless otherwiseindicated. Further many of the groups defined herein can be optionallysubstituted. The listing of substituents in the definition is exemplaryand is not to be construed to limit the substituents defined elsewherein the specification.

Certain of the compounds described herein contain one or more asymmetriccenters and can thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that can be defined, in terms of absolutestereochemistry, as (R)- or (S)-. Unless otherwise specified, thepresent chemical entities, pharmaceutical compositions and methods aremeant to include all such possible isomers, including racemic mixtures,optically pure forms and intermediate mixtures. For the instance,non-limiting example of intermediate mixtures include a mixture of R:Sor S:R isomers in a ratio of 10:90, 13:87, 17:83, 20:80, or 22:78.Optically active (R)- and (S)-isomers can be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.

The term “tautomers” refers to compounds, which are characterized byrelatively easy interconversion of isomeric forms in equilibrium. Theseisomers are intended to be covered by this invention. “Tautomers” arestructurally distinct isomers that interconvert by tautomerization.“Tautomerization” is a form of isomerization and includes prototropic orproton-shift tautomerization, which is considered a subset of acid-basechemistry. “Prototropic tautomerization” or “proton-shifttautomerization” involves the migration of a proton accompanied bychanges in bond order, often the interchange of a single bond with anadjacent double bond. Where tautomerization is possible (e.g. insolution), a chemical equilibrium of tautomers can be reached. Anexample of tautomerization is keto-enol tautomerization. A specificexample of keto-enol tautomerization is the interconversion ofpentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Anotherexample of tautomerization is phenol-keto tautomerization. A specificexample of phenol-keto tautomerization is the interconversion ofpyridin-4-ol and pyridin-4(1H)-one tautomers.

The term “prodrug” refers to a compound, which is an inactive precursorof a compound that is converted into its active form in the body bynormal metabolic processes. Prodrug design is discussed generally inHardma, et al. (Eds.), Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion isprovided in Higuchi, et al., Prodrugs as Novel Delivery Systems, Vol.14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press(1987). To illustrate, prodrugs can be converted into apharmacologically active form through hydrolysis of, for example, anester or amide linkage, thereby introducing or exposing a functionalgroup on the resultant product. The prodrugs can be designed to reactwith an endogenous compound to form a water-soluble conjugate thatfurther enhances the pharmacological properties of the compound, forexample, increased circulatory half-life. Alternatively, prodrugs can bedesigned to undergo covalent modification on a functional group with,for example, glucuronic acid, sulfate, glutathione, amino acids, oracetate. The resulting conjugate can be inactivated and excreted in theurine, or rendered more potent than the parent compound. High molecularweight conjugates also can be excreted into the bile, subjected toenzymatic cleavage, and released back into the circulation, therebyeffectively increasing the biological half-life of the originallyadministered compound.

The term “ester” refers to a compound, which is formed by reactionbetween an acid and an alcohol with elimination of water. An ester canbe represented by the general formula RCOOR′ (where R is a drug and R′is a chemical group).

These prodrugs and esters are intended to be covered within the scope ofthis invention.

Additionally the instant invention also includes the compounds whichdiffer only in the presence of one or more isotopically enriched atomsfor example replacement of hydrogen with deuterium or tritium, or thereplacement of a carbon by ¹³C- or ¹⁴C-enriched carbon.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

Pharmaceutically acceptable salts forming part of this invention includesalts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu,Zn, and Mn; salts of organic bases such as N,N′-diacetylethylenediamine,glucamine, triethylamine, choline, hydroxide, dicyclohexylamine,metformin, benzylamine, trialkylamine, and thiamine; chiral bases suchas alkylphenylamine, glycinol, and phenyl glycinol; salts of naturalamino acids such as glycine, alanine, valine, leucine, isoleucine,norleucine, tyrosine, cystine, cysteine, methionine, proline, hydroxyproline, histidine, ornithine, lysine, arginine, and serine; quaternaryammonium salts of the compounds of invention with alkyl halides, alkylsulphates such as MeI and (Me)₂SO₄; non-natural amino acids such asD-isomers or substituted amino acids; guanidine; and substitutedguanidine wherein the substituents are selected from nitro, amino,alkyl, alkenyl, alkynyl, ammonium or substituted ammonium salts andaluminum salts. Salts may include acid addition salts where appropriatewhich may be sulphates, nitrates, phosphates, perchlorates, borates,hydrohalides, acetates, tartrates, maleates, citrates, fumarates,succinates, palmoates, methanesulphonates, benzoates, salicylates,benzenesulfonates, ascorbates, glycerophosphates, and ketoglutarates.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

The following abbreviations and terms have the indicated meaningsthroughout: PI3-K=Phosphoinositide 3-kinase; PI=phosphatidylinositol;AIDS=Acquired Immuno Deficiency Syndrome; HIV=Human ImmunodeficiencyVirus; MeI=Methyl Iodide; ND: Not determined.

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

The term “cell proliferation” refers to a phenomenon by which the cellnumber has changed as a result of division. This term also encompassescell growth by which the cell morphology has changed (e.g., increased insize) consistent with a proliferative signal.

The terms “co-administration,” “administered in combination with,” andtheir grammatical equivalents, as used herein, encompass administrationof two or more agents to an animal so that both agents and/or theirmetabolites are present in the animal at the same time.Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which both agentsare present.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended application (in vitro or in vivo), orthe subject and disease condition being treated, e.g., the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g. reduction of plateletadhesion and/or cell migration. The specific dose will vary depending onthe particular compounds chosen, the dosing regimen to be followed,whether it is administered in combination with other compounds, timingof administration, the tissue to which it is administered, and thephysical delivery system in which it is carried.

As used herein, “treatment,” “treating,” or “ameliorating” are usedinterchangeably. These terms refers to an approach for obtainingbeneficial or desired results including but, not limited to, therapeuticbenefit and/or a prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated.Also, a therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thepatient, notwithstanding that the patient may still be afflicted withthe underlying disorder. For prophylactic benefit, the compositions maybe administered to a patient at risk of developing a particular disease,or to a patient reporting one or more of the physiological symptoms of adisease, even though a diagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The term “subject” or “patient” refers to an animal (e.g., a dog, cat,horse, or pig), such as a mammal, for example a human. The methodsdescribed herein can be useful in both human therapeutics and veterinaryapplications. In some embodiments, the patient is a mammal, and in someembodiments, the patient is human.

“Radiation therapy” means exposing a patient, using routine methods andcompositions known to the practitioner, to radiation emitters such asalpha-particle emitting radionuclides (e.g., actinium and thoriumradionuclides), low linear energy transfer (LET) radiation emitters(i.e. beta emitters), conversion electron emitters (e.g. strontium-89and samarium-153-EDTMP), or high-energy radiation, including, withoutlimitation, x-rays, gamma rays, and neutrons.

“Signal transduction” is a process during which stimulatory orinhibitory signals are transmitted into and within a cell to elicit anintracellular response. A modulator of a signal transduction pathwayrefers to a compound which modulates the activity of one or morecellular proteins mapped to the same specific signal transductionpathway. A modulator may augment (agonist) or suppress (antagonist) theactivity of a signaling molecule.

The term “selective inhibition” or “selectively inhibit” as applied to abiologically active agent refers to the agent's ability to selectivelyreduce the target signaling activity as compared to off-target signalingactivity, via direct or indirect interaction with the target.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes, but is not limited to, any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, one or more suitablediluents, fillers, salts, disintegrants, binders, lubricants, glidants,wetting agents, controlled release matrices, colorants/flavoring,carriers, excipients, buffers, stabilizers, solubilizers, andcombinations thereof. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions of the invention is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

In certain embodiments, one or more of the compounds described hereinbind specifically to a PI3 kinase or a protein kinase selected from thegroup consisting of mTor, DNA-dependent protein kinase (Pubmed proteinaccession number (PPAN) AAA79184), AbI tyrosine kinase (CAA52387),Bcr-Abl, hemopoietic cell kinase (PPAN CAI19695), Src (PPAN CAA24495),vascular endothelial growth factor receptor 2 (PPAN ABB82619), epidermalgrowth factor receptor (PPAN AG43241), EPH receptor B4 (PPAN EAL23820),stem cell factor receptor (PPAN AAF22141), Tyrosine-protein kinasereceptor TIE-2 (PPAN Q02858), fms-related tyrosine kinase 3 (PPANNP_004110), platelet-derived growth factor receptor alpha (PPANNP_990080), RET (PPAN CAA73131), and any other related protein kinases,as well as any functional mutants thereof.

In other embodiments, the IC₅₀ of a compound described herein for pi10α, pi 10β, pi 10γ, or pi 10δ is less than about 1 μM, less than about100 nM, less than about 50 nM, less than about 10 nM, less than 1 nM orless than about 0.5 nM. In some embodiments, the IC₅₀ of a compounddescribed herein for mTor is less than about 1 μM, less than about 100nM, less than about 50 nM, less than about 10 nM, less than 1 nM or lessthan about 0.5 nM. In some other embodiments, one or more of thecompounds described herein exhibit dual binding specificity and arecapable of inhibiting a PI3 kinase (e.g., a class I PI3 kinase) as wellas a protein kinase (e.g., mTor) with an IC₅₀ value less than about 1μM, less than about 100 nM, less than about 50 nM, less than about 10nM, less than 1 nM or less than about 0.5 nM.

In additional embodiments, the compounds of the present inventionexhibit one or more functional characteristics disclosed herein. Forexample, one or more of the compounds described herein bind specificallyto a PI3 kinase. In some embodiments, the IC₅₀ of a compound describedherein for pi 10α, pi 10β, pi 10γ, or pi 10δ is less than about 1 μM,less than about 100 nM, less than about 50 nM, less than about 10 nM,less than about 1 nM, less than about 0.5 nM, less than about 100 pM, orless than about 50 pM.

In other embodiments, the compounds of the present invention selectivelyinhibit one or more members of type I or class I phosphatidylinositol3-kinases (PI3-kinase) with an IC₅₀ value of about 100 nM or less, about50 nM or less, about 10 nM or less, about 5 nM or less, about 100 pM orless, about 10 pM or less, or about 1 pM or less as measured in an invitro kinase assay.

In yet another aspect, an inhibitor that selectively inhibits one ormore members of type I PI3-kinases, or an inhibitor that selectivelyinhibits one or more type I PI3-kinase mediated signaling pathways,alternatively can be understood to refer to a compound that exhibits a50% inhibitory concentration (IC₅₀) with respect to a given type IPI3-kinase, that is at least 10-fold lower, at least 20-fold lower, atleast 50-fold lower, at least 100-fold lower, at least 1000-fold lowerthan the inhibitor's IC₅₀ with respect to the rest of the other type IPI3-kinases.

As used herein, the term “dual PI3-kinase δ/γ inhibitor” and “dualPI3-kinase δ/γ selective inhibitor” refers to a compound that inhibitsthe activity of both the PI3-kinase δ and γ isozyme more effectivelythan other isozymes of the PI3K family. A dual PI3-kinase δ/γ inhibitoris therefore more selective for PI3-kinase δ and γ than conventionalPI3K inhibitors such as CAL-130, wortmannin and LY294002, which arenonselective PI3K inhibitors.

Inhibition of PI3-kinase δ and γ may be of therapeutic benefit intreatment of various conditions, e.g., conditions characterized by aninflammatory response including, but not limited to, autoimmunediseases, allergic diseases, and arthritic diseases. Importantly,inhibition of PI3-kinase δ and γ function does not appear to affectbiological functions such as viability and fertility.

“Inflammatory response” as used herein is characterized by redness,heat, swelling and pain (i.e., inflammation) and typically involvestissue injury or destruction. An inflammatory response is usually alocalized, protective response elicited by injury or destruction oftissues, which serves to destroy, dilute or wall off (sequester) boththe injurious agent and the injured tissue. Inflammatory responses arenotably associated with the influx of leukocytes and/or leukocyte (e.g.,neutrophil) chemotaxis. Inflammatory responses may result from infectionwith pathogenic organisms and viruses, noninfectious means such astrauma or reperfusion following myocardial infarction or stroke, immuneresponses to foreign antigens, and autoimmune diseases. Inflammatoryresponses amenable to treatment with the methods and compounds accordingto the invention encompass conditions associated with reactions of thespecific defense system as well as conditions associated with reactionsof the non-specific defense system.

The therapeutic methods of the invention include methods for theamelioration of conditions associated with inflammatory cell activation.“Inflammatory cell activation” refers to the induction by a stimulus(including but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including but not limited to,major histocompatibility antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (polymorphonuclear leukocytesincluding neutrophils, basophils, and eosinophils) mast cells, dendriticcells, Langerhans cells, and endothelial cells). It will be appreciatedby persons skilled in the art that the activation of one or acombination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory condition.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

“Transplant rejection” as used herein refers-to any immune responsedirected against grafted tissue (including organs or cells (e.g., bonemarrow), characterized by a loss of function of the grafted andsurrounding tissues, pain, swelling, leukocytosis, andthrombocytopenia).

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy.

“Arthritic disease” as used herein refers to any disease that ischaracterized by inflammatory lesions of the joints attributable to avariety of etiologies.

“Dermatitis” as used herein refers to any of a large family of diseasesof the skin that are characterized by inflammation of the skinattributable to a variety of etiologies.

As previously described, the term “dual PI3-kinase δ/γ selectiveinhibitor” generally refers to a compound that inhibits the activity ofthe PI3-kinase δ and γ isozyme more effectively than other isozymes ofthe PI3K family. The relative efficacies of compounds as inhibitors ofan enzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. IC₅₀ determinations can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Accordingly, a dual PI3-kinase δ/γ selective inhibitor alternatively canbe understood to refer to a compound that exhibits a 50% inhibitoryconcentration (IC₅₀) with respect to PI3-kinase δ and γ, that is atleast 10-fold lower, at least 20-fold lower, or at least 30-fold lowerthan the IC₅₀ value with respect to any or all of the other class I PI3Kfamily members. In an alternative embodiment of the invention, the termdual PI3-kinase δ/γ selective inhibitor can be understood to refer to acompound that exhibits an IC₅₀ with respect to PI3-kinase δ and γ thatis at least 30-fold lower, at least 50-fold lower, at least 100-foldlower, at least 200-fold lower, or at least 500-fold lower than the IC₅₀with respect to any or all of the other PI3K class I family members. Adual PI3-kinase δ/γ selective inhibitor is typically administered in anamount such that it selectively inhibits both PI3-kinase δ and γactivity, as described above.

In certain embodiments, the compounds of the present invention exhibitPI3-kinase δ and γ inhibition almost equally (˜1:1) or at a maximumratio of 1:5, i.e., the compound the of the present invention exhibitalmost equal IC₅₀ values for both PI3-kinase δ and γ enzyme, or at mosta 3 to 8 fold difference between the two.

The methods of the invention may be applied to cell populations in vivoor ex vivo. “In vivo” means within a living individual, as within ananimal or human or in a subject's body. In this context, the methods ofthe invention may be used therapeutically or prophylactically in anindividual. “Ex vivo” or “in vitro” means outside of a livingindividual. Examples of ex vivo cell populations include in vitro cellcultures and biological samples including but not limited to fluid ortissue samples obtained from individuals. Such samples may be obtainedby methods known in the art. Exemplary biological fluid samples includeblood, cerebrospinal fluid, urine, and saliva. Exemplary tissue samplesinclude tumors and biopsies thereof. In this context, the invention maybe used for a variety of purposes, including therapeutic andexperimental purposes. For example, the invention may be used ex vivo orin vitro to determine the optimal schedule and/or dosing ofadministration of a PI3-kinase δ selective inhibitor for a givenindication, cell type, individual, and other parameters. Informationgleaned from such use may be used for experimental or diagnosticpurposes or in the clinic to set protocols for in vivo treatment. Otherex vivo uses for which the invention may be suited are described belowor will become apparent to those skilled in the art.

The compounds of the present invention can be prepared by methods knownin the art, such as those described in International Publication Nos. WO2011/055215, WO 2012/151525, and WO 2013/164801, all of which are herebyincorporated by reference.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising one ormore compounds of the present invention and one or more pharmaceuticallyacceptable carriers or excipients. In one embodiment, the pharmaceuticalcomposition includes a therapeutically effective amount of a compound ofthe present invention. The pharmaceutical composition may include one ormore additional active ingredients as described herein.

The pharmaceutical carriers and/or excipients may be selected fromdiluents, fillers, salts, disintegrants, binders, lubricants, glidants,wetting agents, controlled release matrices, colorants, flavorings,buffers, stabilizers, solubilizers, and combinations thereof.

In one embodiment, the pharmaceutical compositions described hereincontain from about 0.1 mg to about 1,000 mg, such as from about 1 mg toabout 1,000 mg or from about 20 mg to about 800 mg or 50 mg to about 600mg or 50 mg to about 600 mg of one or more compounds of the presentinvention. 100 mg to about 400 mg of one or more compounds of thepresent invention.

The pharmaceutical compositions of the present invention can beadministered alone or in combination with one or more other activeagents. Where desired, the subject compounds and other agent(s) may bemixed into a preparation or both components may be formulated intoseparate preparations to use them in combination separately or at thesame time.

The compounds and pharmaceutical compositions of the present inventioncan be administered by any route that enables delivery of the compoundsto the site of action, such as orally, intranasally, topically (e.g.,transdermally), intraduodenally, parenterally (including intravenously,intraarterially, intramuscularally, intravascularally, intraperitoneallyor by injection or infusion), intradermally, by intramammary,intrathecally, intraocularly, retrobulbarly, intrapulmonary (e.g.,aerosolized drugs) or subcutaneously (including depot administration forlong term release e.g., embedded-under the-splenic capsule, brain, or inthe cornea), sublingually, anally, rectally, vaginally, or by surgicalimplantation (e.g., embedded under the splenic capsule, brain, or in thecornea).

The compositions can be administered in solid, semi-solid, liquid orgaseous form, or may be in dried powder, such as lyophilized form. Thepharmaceutical compositions can be packaged in forms convenient fordelivery, including, for example, solid dosage forms such as capsules,sachets, cachets, gelatins, papers, tablets, suppositories, pellets,pills, troches, and lozenges. The type of packaging will generallydepend on the desired route of administration. Implantable sustainedrelease formulations are also contemplated, as are transdermalformulations.

Methods of Treatment

The amount of the compound to be administered is dependent on the mammalbeing treated, the severity of the disorder or condition, the rate ofadministration, the disposition of the compound and the discretion ofthe prescribing physician. However, an effective dosage is in the rangeof about 0.001 to about 100 mg/kg body weight per day, preferably about1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human,this would amount to about 0.05 to 7 g/day, preferably about 0.05 toabout 2.5 g/day An effective amount of a compound of the invention maybe administered in either single or multiple doses (e.g., twice or threetimes a day).

The compounds of the present invention may be used in combination withone or more of anti-cancer agents (e.g., chemotherapeutic agents),therapeutic antibodies, and radiation treatment.

The compounds of the invention may be formulated or administered inconjunction with other agents that act to relieve the symptoms ofinflammatory conditions such as encephalomyelitis, asthma, and the otherdiseases described herein. These agents include non-steroidalanti-inflammatory drugs (NSAIDs).

EXAMPLES

The examples and preparations provided below further illustrate andexemplify the compounds of the present invention and methods ofpreparing such compounds. It is to be understood that the scope of thepresent invention is not limited in any way by the scope of thefollowing examples and preparations. In the following examples moleculeswith a single chiral center, unless otherwise noted, exist as a racemicmixture. Those molecules with two or more chiral centers, unlessotherwise noted, exist as a racemic mixture of diastereomers. Singleenantiomers/diastereomers may be obtained by methods known to thoseskilled in the art.

As used herein, superscript 1 refers to International Publication No. WO11/055,215 and superscript 2 refers to International Publication No. WO12/151,525. These references describe how various intermediates areprepared.

INTERMEDIATES Intermediate 1:3-(3-fluorophenyl)-2-(1-hydroxypropyl)-4H-chromen-4-one

To a solution of 2-(1-bromopropyl)-3-(3-fluorophenyl)-4H-chromen-4-one¹(8.80 g, 24.36 mmol) in DMSO (85 ml), n-butanol (5 ml) was added andheated to 120° C. for 3 h. The reaction mixture was cooled to roomtemperature (RT), quenched with water and extracted with ethyl acetate.The organic layer was dried over sodium sulphate and concentrated underreduced pressure. The crude product was purified by columnchromatography with ethyl acetate:petroleum ether to afford the titlecompound as a yellow solid (2.10 g, 29%) which was used without furtherpurification in next step.

Intermediate 2: 3-(3-fluorophenyl)-2-propionyl-4H-chromen-4-one

DMSO (1.90 ml, 26.82 mmol) was added to dichloromethane (70 ml) andcooled to −78° C. Oxalyl chloride (1.14 ml, 13.41 mmol) was then added.After 10 minutes, intermediate 1 (2.00 g, 6.70 mmol) in dichloromethane(20 ml) was added dropwise and stirred for 20 min. Triethylamine (7 ml)was added and stirred for 1 h. The reaction mixture was quenched withwater and extracted with dichloromethane. The organic layer was driedover sodium sulphate and concentrated under reduced pressure. The crudeproduct was purified by column chromatography with ethylacetate:petroleum ether to afford the title compound as a yellow liquid(1.20 g, 60%) which was used as such in next step.

Intermediate 3:(+)/(−)-3-(3-fluorophenyl)-2-(1-hydroxypropyl)-4H-chromen-4-one

To a solution of intermediate 2 (0.600 g, 2.02 mmol) in DMF (7.65 ml)under nitrogen purging, formic acid:trietylamine 5:2 azeotrope (1.80 ml)was added followed by [(S,S)tethTsDpenRuCl] (3.0 mg). The reactionmixture was heated at 80° C. for 1.5 hours under continuous nitrogenpurging. The reaction mixture was quenched with water, extracted withethyl acetate, dried over sodium sulphate and concentrated. The crudeproduct was purified by column chromatography with ethylacetate:petroleum ether to afford the title compound as a yellow solid(0.450 g, 74%). Mass: 299.0 (M⁺). Enantiomeric excess: 78%, enriched inthe late eluting isomer (retention time: 9.72 min.) as determined byHPLC on a chiralpak AD-H column.

Intermediate 4:(+)/(−)-3-(3-fluorophenyl)-2-(1-hydroxypropyl)-4H-chromen-4-one

The title compound was obtained as yellow solid (0.500 g, 83%) by usinga procedure similar to the one described for intermediate 3, usingintermediate 2 (0.600 g, 2.02 mmol), DMF (7.65 ml), formicacid:trietylamine 5:2 azeotrope (1.80 ml) and [(R,R)tethTsDpenRuCl] (3.0mg). Mass: 298.9 (M⁺). Enantiomeric excess: 74.8%, enriched in the fasteluting isomer (retention time: 8.52 min.) as determined by HPLC on achiralpak AD-H column.

Intermediate 5:(R)-3-(3-fluorophenyl)-2-(1-hydroxypropyl)-4H-chromen-4-one

Step 1: (R)-2-(1-(benzyloxy)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one:To 2-(3-fluorophenyl)-1-(2-hydroxyphenyl)ethanone (2.15 g, 9.36 mmol),in dichloromethane (20 ml), HATU (4.27 g, 11.23 mmol),R-(+)2-benzyloxybutyric acid (2.00 g, 10.29 mmol) were added and stirredfor 10 min, then triethylamine (14.0 ml, 101.1 mmol) was added dropwiseand stirred at RT for 24 h. The reaction mixture was quenched withwater, extracted with dichloromethane, dried over sodium sulphate andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography with ethyl acetate:petroleum ether to afford thetitle compound as yellow solid (1.65 g, 45%). ¹H-NMR (δ ppm, CDCl₃, 400MHz): 8.24 (dd, J=7.9, 1.5 Hz, 1H), 7.74 (dt, J=7.1, 1.7 Hz, 1H), 7.58(dd, J=8.3, 0.4 Hz, 1H), 7.44-7.06 (m, 10H), 4.51 (d, J=7.8 Hz, 1H),4.34 (d, J=7.8 Hz, 1H), 4.25 (dd, J=7.8, 6.2 Hz, 1H), 2.17-1.90 (m, 2H),0.95 (t, J=7.5 Hz, 3H). Mass: 389.0 (M⁺).

Step 2: (R)-3-(3-fluorophenyl)-2-(1-hydroxypropyl)-4H-chromen-4-one: To(R)-2-(1-(benzyloxy)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (1.50 g,3.86 mmol) in dichloromethane (15 ml) cooled to 0° C. and aluminiumchloride (1.00 g, 7.72 mmol) was added portion wise and stirred at RTfor 6 h. The reaction mixture was quenched with 2N HCl solution,extracted with dichloromethane, dried over sodium sulphate andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography with ethyl acetate:petroleum ether to afford thetitle compound as yellow solid (0.552 g, 48%). ¹H-NMR (δ ppm, CDCl₃, 400MHz): 8.24 (dd, J=8.0, 1.6 Hz, 1H), 7.72 (m, 1H), 7.52 (dd, J=8.4, 0.5Hz, 1H), 7.44 (m, 2H), 7.12-7.01 (m, 3H), 4.49 (t, J=7.0 Hz, 1H), 1.94(m, 2H), 0.93 (t, J=7.5 Hz, 3H). Mass: (299.0 (M⁺). Purity: 96.93%.[α]²⁵ _(D) −14.73 (c=1, CHCl₃). Enantiomeric excess: 85.92%, enriched inthe fast eluting isomer (retention time: 8.57 min.) as determined byHPLC on a chiralpak AS-3R column.

Compound A(RS)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

To a solution of intermediate 1 (2.50 g, 8.41 mmol) in THF (25 ml),tert-butyl 9-trityl-9H-purin-6-ylcarbamate (4.81 g, 10.09 mmol) andtriphenylphosphine (3.31 g, 12.62 mmol) were added and stirred at RT for5 min. Diisopropylazodicarboxylate (2.5 ml, 12.62 mmol) was added andstirred at RT for 2 h. The reaction mixture was concentrated and columnchromatographed with ethyl acetate:petroleum ether to afford a yellowcoloured intermediate. To the intermediate, dichloromethane (65 ml) andtrifluoroacetic acid (7.9 ml) were added and the resulting mixture wasstirred at RT for 12 h. The reaction mixture was then basified withaqueous sodium bicarbonate solution, extracted with dichloromethane anddried over sodium sulphate. The crude product was purified by columnchromatography with methanol:dichloromethane to afford the titlecompound as pale-brown solid (1.05 g, 30%). MP: 148-150° C. Mass: 415.6(M+).

Compound A1(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

Method A:

To a solution of intermediate 3 (0.250 g, 0.838 mmol) in THF (5 ml),tert-butyl 9-trityl-9H-purin-6-ylcarbamate (0.479 g, 1.00 mmol) andtriphenylphosphine (0.329 g, 1.25 mmol) were added and the resultingmixture was stirred at RT for 5 min. Diisopropylazodicarboxylate (0.25ml, 1.25 mmol) was then added and stirred at RT for 12 h. The reactionmixture was concentrated and column chromatographed with ethylacetate:pet. ether to afford the yellow coloured intermediate. To theintermediate in dichloromethane (6 ml), trifluoroacetic acid (1.2 ml)was added stirred at RT for 12 h. The reaction mixture was basified withaqueous sodium bicarbonate solution, extracted with dichloromethane anddried over sodium sulphate. The crude product was purified by columnchromatography with methanol:dichloromethane to afford the titlecompound as an off-white solid (0.015 g, 4%). MP: 137-140° C. ¹H-NMR (δppm, DMSO-d₆, 400 MHz): 12.94 (s, 1H), 8.12-8.10 (m, 4H), 7.84-7.80 (m,1H), 7.61 (d, J=8.3 Hz, 1H), 7.50-7.41 (m, 2H), 7.28-7.18 (m, 3H),5.20-5.06 (m, 1H), 2.10-1.90 (m, 2H), 0.84 (t, J=3.7 Hz, 3H).Enantiomeric excess: 77.4% as determined by HPLC on a chiralpak AD-Hcolumn, enriched in the fast eluting isomer (retention time=7.90 min.).

Method B:

To a solution of intermediate 5 (2.60 g, 8.68 mmol) in THF (52 ml),tert-butyl 9-trityl-9H-purin-6-ylcarbamate (4.96 g, 10.42 mmol) andtriphenylphosphine (2.76 g, 13.03 mmol) were added and the resultingmixture was stirred at RT for 5 min. Diisopropylazodicarboxylate (0.25ml, 1.25 mmol) was then added and stirred at RT for 12 h. The reactionmixture was concentrated and column chromatographed with ethylacetate:petroleum ether to afford the yellow coloured intermediate. Tothe intermediate in dichloromethane (55 ml), trifluoroacetic acid (14.2ml) was added and stirred at RT for 12 h. The reaction mixture wasbasified with aqueous sodium bicarbonate solution, extracted withdichloromethane and dried over sodium sulphate. The crude product waspurified by column chromatography with methanol:dichloromethane toafford the title compound as pale-yellow solid (1.00 g, 27%). MP:168-170° C. Mass: 416.5 (M⁺+1) Enantiomeric excess: 86.5% as determinedby HPLC on a chiralpak AD-H column, enriched in the fast eluting isomer(retention time=7.90 min.).

Method C:

The title compound was separated by preparative SFC conditions fromCompound A (1.090 g) on a CHIRALPAK AY-H column (250×30 mm; 5 μm) usingmethanol:CO₂ (35:65) as the mobile phase at a flow rate of 80 g/min.Off-white solid (0.378 g). e.e. 100%. Rt: 2.37 min. Mass: 416.1 (M⁺+1).MP: 149-152° C.

Compound A2(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

Method A:

The title compound was obtained as an off-white solid (0.015 g, 4%) byusing a procedure similar to the one described for compound A1 (MethodA) using tert-butyl 9-trityl-9H-purin-6-ylcarbamate (0.479 g, 1.00mmol), intermediate 4 (0.250 g, 0.838 mmol), triphenylphosphine (0.329g, 1.25 mmol), THF (5 ml) and diisopropylazodicarboxylate (0.25 ml, 1.25mmol), followed by the cleavage of the intermediate with trifluoroaceticacid (1.2 ml) and dichloromethane (6 ml). MP: 139-141° C. Mass: 415.6(M+). Enantiomeric excess: 81.6% as determined by HPLC on a chiralpakAD-H column, enriched in the late eluting isomer (retention time=10.81min.).

Method B:

The title compound was separated by preparative SFC conditions fromCompound A (1.090 g) on a CHIRALPAK AY-H column (250×30 mm; 5 μm) usingmethanol:CO₂ (35:65) as the mobile phase at a flow rate of 80 g/min.Off-white solid (0.434 g). e.e. 98%. Rt: 3.71 min. Mass: 416.1 (M⁺+1).MP: 162-164° C.

BIOLOGICAL ASSAYS

The pharmacological properties of the compounds described herein may beconfirmed by a number of pharmacological assays. The pharmacologicalassays which have been carried out with the compounds according to theinvention and/or their pharmaceutically acceptable salts are exemplifiedbelow

Assay 1: Fluorescent Determination of PI3 Kinase Enzyme Activity

Phosphoinositide 3 kinases (PI3K) belong to a class of lipid kinasesthat play a critical role in the regulation of several key cellularprocesses. The PI3K are capable of phosphorylating the 3-hydroxyposition of phosphoinositols thereby generating second messengersinvolved in downstream signalling events. The homogenous time resolvedfluorescence (HTRF) assay allows detection of 3,4,5-triphosphate (PIP3)formed as a result of phosphorylation of phosphotidylinositol4,5-biphosphate (PIP2) by PI3K isoforms such as α, β, γ or δ.

PI3K isoform activity for α, β, γ or δ was determined using a PI3K humanHTRF™ Assay Kit (Millipore, Billerica, Mass.) with modifications. Allincubations were carried out at room temperature. Briefly, 0.5 μl of 40×inhibitor (in 100% DMSO) or 100% DMSO were added to each well of a384-well white plate (Greiner Bio-One, Monroe, N.C.) containing 14.5 μl1× reaction buffer/PIP2 (10 mM MgCl₂, 5 mM DTT, 1.38 μM PIP2) mix withor without enzyme, followed by 5 μl/well of 400 μM ATP and incubated foran additional 30 minutes. Reaction was terminated by adding 5 μl/wellstop solution (Millipore, Billerica, Mass.). 5 μl of detection mix(Millipore, Billerica, Mass.) were then added to each well and wasincubated for 6-18 hours in the dark. HRTF ratio was measured on amicroplate reader (BMG Labtech., Germany) at an excitation wavelength of337 nm and emission wavelengths of 665 and 615 nm with an integrationtime of 400 msec counting delay of 50 msec. The results for Compounds A1and A2 are shown in Table 1 below. Comparative data for Compound A1 andExample 47 of WO 11/055,215 are provided in Table 2.

TABLE 1 IC₅₀ (nM) Compound Pi3Kδ Pi3Kα Pi3Kβ Pi3Kγ A1 23.85 >10000 >400024.05 A2 >10 μM ND ND >10 μM

TABLE 2 PI3Kδ PI3Kγ Compound IC₅₀ in nM % Inhibition at 1 μm IC₅₀ in nMExample 47 of 105.9 25.54 ND WO 11/055215 Compound A1 23.85 — 24.05Assay 2: In Vitro Cell Proliferation Assay in Leukemic Cell Lines

Growth inhibition assays were carried out using 10% FBS supplementedmedia. Cells were seeded at a concentration of 5000-20,000 cells/well ina 96-well plate. Test compounds at a concentration range from 0.01 to10000 nM were added after 24 h. Growth was assessed using the3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dyereduction test at 0 h (prior to the addition of the test compound) and72 h after the addition of test compound. Absorbance was read on aFluostar Optima (BMG Labtech, Germany) at a wave length of 450 nm. Datawere analysed using GraphPad Prism and percent inhibition due to thetest compound compared to the control was calculated accordingly.

Compound A1 caused a reduction in T-lymphoma (MOLT-4, Jurkat, CCRF-CEM,Hut-78 & HuT-102) cell viability with GI₅₀ values ranging from 1-5 μMfor the dose range tested. Additionally, the compound did not displayany apparent cytotoxicity over the 72-h incubation period up to 10 μM.

Assay 3: Inhibition of AKT Phosphorylation in Leukemic Cell Lines

MOLT-4, Jurkat, CCRF-CEM, Hut-78, HuT-102, Sez4 and HH cells wereincubated with desired concentrations of compound for 48 h. Cells werelysed and pAKT determined by Western Blotting. Bands were quantifiedusing ImageJ and normalized to actin.

Compound A1 caused a reduction in pAKT expression in T-lymphoma (MOLT-4,Jurkat, CCRF-CEM, Hut-78 & HuT-102) cell lines with EC50 values rangingfrom 0.5-2 μM for the dose range tested. The results are shown in FIG.8.

Assay 4: Inhibition of PI3K δ and γ Signalling in Basophils from HumanWhole Blood

PI3K δ and γ signalling in basophils manifested by an alteration ofanti-FcεR1 or fMLP induced CD63 expression is a useful pharmacodynamicmarker determined using the Flow2CAST® kit (Buhlmann Laboratories,Switzerland). Briefly, it involves the following steps:

-   -   Mix the anti-coagulated blood sample by inverting the        venipuncture tube several times;    -   Prepare fresh and pyrogen-free 3.5 ml polypropylene or        polystyrene tubes suitable for Flow Cytometry measurements;    -   Add 49 μl of patient's whole blood to each tube;    -   Add 1 μl of 10% DMSO (background) or compound (10% DMSO) to the        assigned tubes and mix gently. Incubate at room temperature for        15 minutes;    -   Pipet 50 μl of the Stimulation buffer (background) or anti-FcεRI        Ab or fMLP to each tube;    -   Add 100 μl of Stimulation Buffer to each tube;    -   Mix gently. Add 20 μl Staining Reagent (1:1 mix of FITC-CD63 and        PE-CCR3) to each tube;    -   Mix gently, cover the tubes and incubate for 15 minutes at        37° C. in a water bath. (using an incubator will take about 10        minutes longer incubation time due to less efficient heat        transfer);    -   Add 2 ml pre-warmed (18-28° C.) Lysing Reagent to each tube, mix        gently;    -   Incubate for 5-10 minutes at 18-28° C.;    -   Centrifuge the tubes for 5 minutes at 500×g;    -   Decant the supernatant by using blotting paper;    -   Resuspend the cell pellet with 300-800 μl of Wash Buffer; and    -   Vortex gently and acquire the data on the flow cytometer within        the same day.

Percent CD63 positive cells within the gated basophil population weredetermined in different treatment groups and normalized to vehiclecontrol.

Compound A1 exhibited a EC₅₀ of <40 nM for FcεR1 (PI3K δ) and a IC₅₀ of<40 nM for fMLP (PI3K γ) (n=10). The results are shown in FIG. 9.

Assay 4a: Cellular Activity Demonstrating Selectivity of Compound A1Towards PI3K Delta and PI3K Gamma Isoform

Assay 4A1: Anti-IgM Induced B-Cell Proliferation (for PI3Kδ Selectivity)

The objective of this study was to assess the inhibitory potential ofCompound A1 on anti-IgM induced human B-cell proliferation.

Plating and Treatment

-   -   Isolated B-cells were re-suspended to 1.0×10⁶ cells per ml. 100        μl of cell suspension was added to each well of a 96-well plate.        Triplicates were maintained.    -   50 μl of drug dilution was added and mixed well. A DMSO blank        and inducer blank were maintained.    -   Treated plate was incubated for 30 min at 37° C., 5% CO₂ and        then 50 μl of 4× inducer was added and mixed by pipetting.    -   Plate was incubated at 37° C., 5% CO₂ for 72 h.    -   Media was aspirated and 150 μl of DMSO was added to dissolve the        formazan crystals.    -   Absorbance was read at A₅₆₀ and A₆₄₀ nm.

The data demonstrates the inhibitory potential of Compound A1 on PI3Kδmediated induction of human B-cell proliferation. See, e.g., Baeker etal. Journal of Immunology, 134: 3532-3538, 1985.

Assay 4A2: LPA Induced AktS473 Phosphorylation in 3T3 Fibroblasts (forPI3Kβ Selectivity)

The objective of this study was to determine the effect of Compound A1on PI3Kβ kinase mediated LPA induced AktS473 phosphorylation in 3T3fibroblasts.

-   -   3T3 cells were treated with desired concentrations of the test        compound for 15 min. 1 ml of 2×LPA was added such that the final        concentration was 5 μM and incubated for 5 min.    -   Media was discarded and washed with 1 ml of ice-cold 1×PBS.    -   250 μl of cell lysis buffer was added and incubated on ice for        30 min.    -   Samples were centrifuged and supernatant was at −80° C. until        analysis.    -   Samples were analyzed by Western Blotting using pAKT (S473) as        the primary and anti-rabbit IgG-HRP as a secondary antibody.    -   Intensity of the bands was determined using ImageJ 1.42q (NIH,        USA) and normalized to Actin (loading control). Data was plotted        using GraphPad Prism (Version 5.02).

The results demonstrate the selectivity of Compound A1 over the betaisoform of PI3K. See Albuquerque et al., J. Biol. Chem. 278,39830-39838, 2003.

Assay 4A3: c5a Induced AktS473 Phosphorylation in RAW 264.7 Macrophages(for PI3Kγ Selectivity)

The objective of this study was to determine the effect of Compound A1on PI3Kγ kinase mediated c5a induced AktS473 phosphorylation in RAW264.7 macrophages.

-   -   RAW 264.7 cells were treated with desired concentrations of the        test compound for 15 min. 1 ml of 2×c5a was added such that the        final concentration was 50 ng/ml and incubated for 15 min.    -   Media was discarded and washed with 1 ml of ice-cold 1×PBS.    -   250 μl of cell lysis buffer was added and incubated on ice for        30 min.    -   Samples were centrifuged and supernatant was stored at −80° C.        until analysis    -   Samples were analyzed by Western Blotting using pAKT (S473) as        the primary and anti-rabbit IgG-HRP as a secondary antibody.    -   Intensity of the bands was determined using ImageJ 1.42q (NIH,        USA) and normalized to Actin (loading control). Data was plotted        using GraphPad Prism (Version 5.02).

Inhibition of pAktS473, a downstream marker of PI3Kδ signaling suggestsa role for Compound A1 in the oncogenic pathways regulated by Akt in c5ainduced RAW 264.7 cells. See To et al. Am. J. Respir. Crit. Care Med.,182, 897-904, 2010.

Assay 4A4: PDGF Induced Akt Phosphorylation in 3T3 Cells (for PI3K αSelectivity)

The objective of this study was to determine the effect of Compound A1on PI3Kα kinase mediated AktS473 phosphorylation in PDGF induced 3T3fibroblasts.

-   -   3T3 cells were treated with desired concentrations of the test        compound for 15 min. 1 ml of 2×PDGF was added such that the        final concentration was 20 ng/ml and incubated for 10 min.    -   Media was discarded and washed with 1 ml of ice-cold 1×PBS.    -   250 μl of cell lysis buffer was added and incubated on ice for        30 min.    -   Samples were centrifuged and supernatant was collected and        stored at −80° C. until analysis.    -   Samples were analyzed by Western Blotting using pAKT (S473) as        the primary and anti-rabbit IgG-HRP as a secondary antibody.    -   Intensity of the bands was determined using ImageJ 1.42q (NIH,        USA) and normalized to Actin (loading control). Data was plotted        using GraphPad Prism (Version 5.02).

No inhibition was observed at 10 μM of Compound A1, demonstrating theselectivity of Compound A1 over the alpha isoform of PI3K. SeeAlbuquerque et al., J. Biol. Chem. 278, 39830-39838, 2003.

The Table below summarizes the results from Assays 4A1-4A4.

CELLULAR ACTIVITY DEMONSTRATING SELECTIVITY OF COMPOUND A1 TOWARDS PI3KDELTA AND PI3K GAMMA ISOFORM Cellular IC₅₀ PI3K alpha (PDGF induced pAKTin >10000 nM 3T3 fibroblasts) Cellular IC₅₀ PI3K beta (LPA induced pAKTin 2067 nM 3T3 fibroblasts) Cellular IC₅₀ PI3K delta (anti-IgM inducedhuman 38.1 nM B-cell proliferation) Cellular IC₅₀ PI3K gamma (c5ainduced pAKT in 22.3 nM RAW macrophages)Assay 5: Inhibition of Apoptosis in Leukemic Cell Lines

Apoptosis in leukemic cells was determined using an in-situ Caspase 3kit (Millipore, US) as outlined below:

-   -   Seed leukemic cells—at a density of 1×10⁶ cells/well in a 6 well        plate    -   Add test compound/DMSO at desired concentrations    -   Incubate the plate for 24 hrs at 37° C. in 5% CO₂ incubator    -   Collect cells in a 2 ml centrifuge tube    -   Add 1.6 μL of freshly prepared 5×FLICA reagent and mix cells by        slightly flicking the tubes    -   Incubate tubes for 1 hour at 37° C. under 5% CO₂    -   Add 2 ml of 1× wash buffer to each tube and mix    -   Centrifuge cells at <400×g for 5 minutes at room temperature.    -   Carefully remove and discard supernatant, and gently vortex cell        pellet to disrupt any cell-to-cell clumping.    -   Resuspend cell pellet in 300 ul of 1× wash buffer    -   Place 100 μL of each cell suspension into each of two wells of a        black microtiter plate. Avoid creation of bubbles.    -   Read absorbance of each microwell using an excitation wavelength        of 490 nm and an emission wavelength of 520 nm    -   Percent increase in caspase-3 activity manifested by an increase        in fluorescence compared to the control blank is to be        calculated.

Compound A1 caused a dose-dependent induction in caspase-3 activity inT-lymphoma (MOLT-4, Jurkat, CCRF-CEM, Hut-78 & HuT-102) cell lines.

Assay 6: Screening for pAKT Inhibition in Human Primary CTCL Cells

Flow cytometry analysis of pAKT inhibition: Purified malignant T cellswere isolated from donors and cultured overnight in RPMI/1% BSA. Cellswere incubated with the test compound for 1.5 hr with a cytokine mixadded for the final 30 minutes. The composition of the cytokine mix was20 ng/ml IL2+5 ng/ml IL7+10 ng/ml IL15+10% FBS. AKT phosphorylation wasdetermined by flow cytometry.

Treatment with Compound A1 caused a dose dependent reduction AKTphosphorylation with EC₅₀ ranging from 40-300 nM (% inhibition data).

Assay 6A: Screening for Anti-Cancer Activity in Human CLL Cells

Primary CLL cells were enriched using Rosette-Sep B cells from Stem CellTechnology, generally giving purity of >97% of B cells/CLL cells. Cellswere seeded at 2.5×10⁵ per well in a 96 well with either serum freemedium (SFM) or SFM+10% heat-inactivated fetal bovine serum (volume 100microliters) in the presence of desired concentrations of the testcompound and cultured for 3 days at 37° C. in a carbon dioxideincubator. Cytotoxicity was determined using the MTS assay.

Compound A1 induces cytotoxicity in CLL cells with a median EC₅₀ of <100nM in serum-free and <700 nM in 10% FBS media.

Assay 6B: Screening for Anticancer Activity in Patient DerivedB-Lymphoma Cells

Primary cells from lymphoid tumors were exposed to the test compound[Compound A1] to assess the induction of cell death. Cells were derivedfrom diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),splenic marginal zone lymphoma (SMZL), extranodal marginal zone lymphoma(EMZL), or chronic lymphocytic leukemia (CLL, no.=1). Cells were treatedwith desired concentrations of the compound and apoptosis (Annexin V/PI)was assessed by flow-cytometry after a 48-h incubation period.

Almost all the primary cells derived from B-cell lymphomas underwent anincrease in cell death when exposed to the test compound (Compound A1)at a concentration of 4 μM. The phenomenon appeared more evident amongprimary cells derived from small cell lymphomas (MZL, MCL, and CLL).

Assay 6C: Inhibition of AKT Phosphorylation in B-Lymphoma Cell Lines

LY-1, LY-10, Daudi, JEKO, REC and MAVER cells were incubated withdesired concentrations of [compound A1] for 48 hours. Cells were lysedand pAKT was determined by Western Blotting. Bands were quantified usingImageJ and normalized to actin.

Compound A1 exhibited an EC₅₀ of 20-200 nM across the B-lymphoma celllines tested.

Assay 6D: Cytokine Assay in Anti-Human CD3 and CD28 Vo-StimulatedPrimary T-Cells

The objective of this study was to assess the inhibitory potential ofCompound A1 on cytokines produced by anti-human CD3/CD28 co-stimulatedprimary T-cells.

Plating and Treatment

-   -   Plates were coated with 50 μl of anti-human CD3 at a        concentration of 100 ng/ml either overnight at 4° C. or for 2 h        at 37° C. After incubation, plates were washed twice with        sterile PBS to remove unbound antibody.    -   Isolated human T-cells were re-suspended to 0.625×10⁶ cells per        ml in 1.5 ml tubes and 1 μl of drug dilution (1000×) was added.        A DMSO blank and un-induced blank were maintained.    -   Cells were incubated with compound at room temperature for 30        min and then added to anti-human CD3 coated wells of a 96-well        plate, 240 μl each. Anti-human CD28 was added immediately, 10 μl        (25×) per well.    -   Plate was incubated at 37° C., 5% CO₂ for 24 h.    -   Plates were centrifuged at 4000 rpm at room temperature,        supernatant was collected and stored at −20° C.    -   Cytokines were determined using a commercial ELISA kit with        absorbance read at 450 and 570 nm.

IC₅₀ values were calculated from eight independent experiments. CompoundA1 inhibited anti-human CD3-CD28 induced T cell cytokines with an IC₅₀of 24, 9.54 and 20.6 nM for TNFα, IFNγ and IL2 respectively. The resultsare shown in FIG. 10.

Assay 7: Lipopolysaccharide Induced Pulmonary Neutrophilia in FemaleWistar Rat Model

An exaggerated recruitment and subsequent activation of neutrophil islikely to be important for the development and course of severalinflammatory diseases in the airways and lungs, such as severe asthma,chronic obstructive pulmonary disease, cystic fibrosis, and acuterespiratory distress syndrome. The mechanisms by which neutrophilcontribute to these diseases may involve the release of proteolyticenzymes, such as neutrophil elastase, and free oxygen radicals. Whenreleased, these compounds can cause bronchoconstriction, bronchialhyperreactivity, hyper-secretion, epithelial damage, and tissueremodelling in the airways.

After the quarantine period, fasted animals were randomized and dividedinto various groups depending on their body weights. The test compound[Compound A1] was prepared as a suspension in a vehicle consisting of0.5% methylcellulose in which Tween 80 as a suspending agent. Thecompound or vehicle was administered by oral gavage in a volume of 10mL/kg. Female Wistar rats were anaesthetized with ketamine and LPSsolution was administered intratracheally one hour after compoundadministration at a dose of 1 mg/kg. 6 h after LPS instillation, animalswere exsanguinated under anaesthesia, and then trachea was cannulatedand the lungs were lavaged with 5-ml aliquots of heparinised PBS (1unit/ml) four times through tracheal cannula (total volume 20 ml).Bronchioalveolar lavage (BAL) fluid was been stored at 2-8° C. untilassayed for total cell and differential leukocyte count.Bronchioalveolar fluid was centrifuged (500×g for 10 min) and theresulting cell pellet was resuspended in 0.5 ml of heparinised saline.The total numbers of white blood cells were determined in BAL fluid orblood by using a blood cell counter and was adjusted to 1×10⁶ cell/ml.Differential cell count was calculated manually. One hundred microlitersof the cell suspension was centrifuged using cytospin 3 to prepare acell smear. The cell smear was stained with a blood staining solutionfor differentiation and slides were microscopically observed to identifyeosinophil according to their morphological characteristics. The numberof each cell type among 300 white blood cells in the cell smear wasdetermined and expressed as a percentage. The number of eosinophil ineach BALf or blood was calculated.

Compound A1 showed a dose-dependent reduction of neutrophil infiltrationinto the lungs with an ED₅₀ of 6.5 mg/kg suggesting a therapeutic rolein inflammatory disorders. The results are shown in FIG. 1.

Assay 8: Lipopolysaccharide-Induced Rat Air Pouch Model of Inflammation

Leukocyte recruitment and the formation of pro-inflammatory mediators,including different cytokines, are the hallmark of an inflammatoryresponse. The air-pouch model was originally developed as a facsimilesynovium for the study of inflammatory processes that occur inrheumatoid arthritis (RA). The model allows the differentialquantification of leukocyte species that accumulate in the air-pouchwall (tissue) as well as those that transmigrate into the air-pouchcavity (lavage), and it allows the characterization of the chemokinesand adhesion molecules responsible for diapedesis induced by a varietyof inflammatory stimuli.

Female Wistar rats (175-200 g) were acclimatized for seven days prior tothe start of the experiment Animals were randomly distributed to variousgroups based on their body weights Animals were anaesthetised with etherand subcutaneous air pouches were made by injecting 20 ml of sterile airunder the skin in the intra-scapular area (day 0) and maintained with asecond 10-ml injection of sterile-filtered air on day 4. On day 6, oraltreatment was commenced 1 h prior to induction of inflammation by s.c.injection of LPS solution on day 6. A volume of 5-ml of LPS solutiondissolved in sterile saline (100 μg/kg) was injected into each pouch.Samples of pouch fluid were taken at 6 h after administration of LPS byflushing the pouch with 5 ml of sterile saline and withdrawing 4 ml offluid. The numbers of leukocytes present in pouch fluid was determinedmicroscopically using a haemocytometer. Differential cell content wasdetermined by microscopic examination of fluid smears stained withDiff-Quik.

Compound A1 caused a dose-dependent reduction of neutrophil migrationinto the rat air pouch with an ED₅₀ of 2.6 mg/kg suggesting atherapeutic role in rheumatoid arthritis. The results are shown in FIG.2.

Assay 9: Lipopolysaccharide Induced TNF-α Production

Fasted female wistar rats were randomized into different groupsdepending on their body weights. Test compound (Compound A1) wasprepared as a suspension in a vehicle consisting of 0.5%methylcellulose. The compound or vehicle was administered by oral gavagein a volume of 10 mL/kg. LPS solution was administered intraperitoneallyone hour after compound administration at a dose of 0.3 mg/kg. Blood wascollected in serum separator tubes via cardiac puncture ninety minutesafter LPS injection. Serum was separated and stored at −20° C. and willbe analysed for TNFα by ELISA.

Compound A1 reduced plasma TNFα concentrations suggesting a therapeuticrole in inflammatory disorders (percent inhibition observed at 1, 3 and10 mg/kg was 5%, 15%, and 40%, respectively). The results are shown inFIG. 3.

Assay 10A: Ovalbumin Induced Pulmonary Eosinophilia in Male Guinea Pigs

Airway inflammation and hyper-responsiveness (AHR) are hallmarks anddistinguishing features of bronchial asthma. Provocation ofpre-sensitized mice with the same allergen induces airway inflammationwith preferential eosinophilic infiltration and, as a consequence, AHR.Pulmonary eosinophilia and airway remodelling in conjunction withaltered neural control of airway tone and airway epithelial desquamationmay contribute to AHR in asthma.

After the quarantine period, 0.3 mL of blood samples was collected fromorbital vein by retro-orbital plexus method from each individual animaland analysed on a cell analyser (ADVIA 2120, Siemens). Based on theirtotal cell count, guinea pigs were randomized and divided into variousgroups. Ear pinna was marked with an indelible marking pen foridentification. On day 0, weights were recorded and animals weresensitized with 50 μg of Ovalbumin (OVA) and 10 mg of alum solution (1mL) intraperitoneally. On day 7 and day 14, the above sensitizationprotocol was repeated. Animals were observed for any signs of illness orreaction to the sensitization up to day 19 and recorded if any. On day19, 20, and 21, after the treatment with test compound by oral gavage,30 mins later animals were exposed to 0.5% w/v, 0.5% and 1% Ovalbuminchallenge respectively. Control & sham group animals were treated with0.5% w/v methyl cellulose (vehicle). Sham control groups were sensitizedwith 10 mg of alum on day 0, 7 & 14 and exposed to saline solution (SAL)with the same nebulization rate on day 19, 20 and 21. Twenty hours afterlast OVA challenge, airway hyperresponsiveness was measured by wholebody plethysmograph against cumulative doses of methacholine challenge(75, 100, 125 & 150 μg/ml), after measuring the airway response, bloodsamples and BAL fluid was collected. Samples were analysed for totalcell count by using neubuear chamber under microscope and differentialleukocyte count was done manually.

As depicted in the FIGS. 4A and 4B, Compound A1 caused a significantdose dependent reduction in airway hyperresponsiveness againstmethacholine challenge of sensitized Guinea pigs.

As depicted in the FIGS. 4C-4E, Compound A1 caused a significant dosedependent reduction in eosinophil infiltration into the bronchioalveolarlavage fluid of sensitized Guinea pigs.

Assay 10B: Murine Asthma Model

Airway inflammation and hyper-responsiveness (AHR) are hallmarks anddistinguishing features of bronchial asthma. Provocation ofpre-sensitized mice with the same allergen induces airway inflammationwith preferential eosinophilic infiltration and, as a consequence, AHR.Pulmonary eosinophilia and airway remodelling in conjunction withaltered neural control of airway tone and airway epithelial desquamationmay contribute to AHR in asthma. After the quarantine period, based ontheir body weights, mice were randomized and divided into four groups(n=7). Tails were marked with an indelible marking pen foridentification. On day 0, weights were recorded and animals weresensitized with 100 μg of Ovalbumin and 10 mg of alum solution (0.2 mL)intraperitoneally. On day 7 and day 14, the above sensitization protocolwas repeated. Animals were observed for any signs of illness or reactionto the sensitization up to day 24 and recorded if any. On day 24, 25,and 26, after the treatment with test compound by oral gavage, 30 minslater animals were exposed to 10% w/v Ovalbumin challenge. Control andsham group animals were treated with 0.5% w/v methyl cellulose(vehicle). Sham control groups were sensitized with 10 mg of alum on day0, 7 & 14 and exposed to saline solution with the same nebulization rateon day 24, 25 & 26. Forty eight hours after last OVA challenge, airwayhyperresponsiveness was measured by whole body plethysmograph againstcumulative doses of methacholine challenge (2.5, 10, 50 and 100 mg/ml),after measuring the airway response, blood samples and BAL fluid wascollected. Samples were analysed for total cell count by using neubuearchamber under microscope and differential leukocyte count was donemanually. As depicted in the FIGS. 5A and 5B, Compound A1 at a dose of 3mg/kg caused a significant reduction in airway hyperresponsivenessagainst methacholine challenge of ovalbumin sensitized mice.

As depicted in the FIGS. 5C-5E, Compound A1 at a dose of 3 mg/kg causeda significant inhibition of eosinophil infiltration into thebronchioalveolar lavage fluid of ovalbumin sensitized mice.

Assay 11: Collagen Induced Arthritis in Lewis Rats

Female wistar rats were acclimatized for seven days prior to the startof the experiment and were randomly distributed to various groups basedon their body weights. On day 0, animals were treated by intradermalinjection of 500 μg of bovine collagen type II emulsified with completeFreund's adjuvant (IFA) containing MTB (4 mg/mL) delivered at the baseof the tail. On day 7 after primary immunization, animals were treatedby booster injection of 300 μg CII in incomplete Freund's adjuvant byintradermal injection at the base of the tail. Onset of arthritis inankle joints usually became visually apparent between days 12 and 14.Animals were treated with test compound or vehicle (orally administered)from the day after onset of arthritis until end of the experiment (day28) as a therapeutic group. Arthritis Scores were taken by visuallyexamination for signs of joint inflammation regularly throughout thestudy period. Body weights and paw volumes, paw thickness has been takenon day 0, 3, 7, 10, 12, 14, 17 21, 24 and 28. On d28, at the end of thestudy, blood has been withdrawn at necropsy and processed to serum orplasma and all joints were taken and both fore paw and hind paws werefixed in 10% formalin for histopathology analysis after taking the smallpiece of tissue from each joint and stored at −80° C. for cytokineanalysis in tissue homogenate. Clinical Scoring Criteria for Fore andHind Paws: 0=normal; 1=one hind or fore paw joint affected or minimaldiffuse erythema and swelling; 2=two hind or fore paw joints affected ormild diffuse erythema and swelling; 3=three hind or fore paw jointsaffected or moderate diffuse erythema and swelling; 4=marked diffuseerythema and swelling, or =four digit joints affected; 5=severe diffuseerythema and severe swelling entire paw, unable to flex digits.

Compound A1 dosed therapeutically in the rat CIA model demonstratessignificant efficacy in reduction of knee and as well as ankle swelling.

Histological analysis of joints at study end demonstrates completestructural preservation at 15 mg/kg Compound A1. For comparison, avehicle-dosed animal shows synovial (S) inflammation and significantevidence of bone resorption, pannus formation, and cartilage degradationand, as depicted in FIGS. 6A-6D, Compound A1 shows significant reductionin individual and summed histopathological scores for both knee andankle.

Assay 12: Acute CSE Induced Cell Infiltration in Male Balb/c Mice

Animals (male Balb/c mice) are to be acclimatized for seven days priorto the start of the experiment Animals are to be randomly distributed tovarious groups based on their body weights. On day 1, mice are to beadministered the test compound or vehicle by oral/intranasal route andafter 1 hr the test compound administration the animals are to beanaesthetised with ether and cigarette smoke extract is to beadministered by intranasal route in volume of 50 μl/mouse and repeatedthe CSE exposure to animals daily after the test compound administrationfor four days (d1 to d4). On day 5, 24 hours after last CSE exposureanimals are to be exsanguinated under anesthesia, and the trachea is tobe cannulated and the lungs are lavaged with 0.5-ml aliquots ofheparinised PBS (1 unit/ml) four times through tracheal cannula (totalvolume 2 ml). BAL stored at 2-8° C. until assayed for total cell anddifferential leukocyte count. Bronchioalveolar fluid is to becentrifuged (500×g for 10 min) and the resulting cell pellet has to beresuspended in 0.5 ml of heparinised saline. The total number of whiteblood cells is to be determined in BAL fluid and blood using a bloodcell counter and adjusted to 1×10⁶ cell/ml. Differential cell count isto be calculated manually. Forty microliters of the cell suspension isto be centrifuged using cytospin 3 to prepare a cell smear. The cellsmear is to be stained with a blood staining solution fordifferentiation and microscopically has to be observed to identifyeosinophil according to their morphological characteristics. The numberof each cell type among 300 white blood cells in the cell smear are tobe determined and to be expressed as a percentage, and the number ofneutrophils and macrophages in each BALf are to be calculated.

Assay 13: Sub-Chronic CSE Induced Cell Infiltration in Male Balb/c Mice

Animals (male Balb/c mice) are to be acclimatized for seven days priorto the start of the experiment Animals are to be randomly distributed tovarious groups based on their body weights. On day 1, animals are to beanaesthetised with ether and cigarette smoke extract is to beadministered by intranasal route in volume of 50 μl/mouse and repeatedthe CSE exposure to animals daily for eight days (d1 to d8). On day 9,mice are to be administered by test compound or vehicle byoral/intranasal route and after 1 hr test compound administrationanimals are to be anaesthetised with ether and cigarette smoke extractis to be administered by intranasal route in volume of 50 μl/mouse andanimals are to be exposed to CSE daily after the test compoundadministration for next three days (d9 to d11), on day 12, twenty fourhours after last CSE exposure animals are to be exsanguinated underanesthesia, and the trachea is to be cannulated and the lungs are to belavaged with 0.5-ml aliquots of heparinised PBS (1 unit/ml) four timesthrough tracheal cannula (total volume 2 ml). BAL stored at 2-8° C.until assayed for total cell and differential leukocyte count.Bronchioalveolar fluid was centrifuged (500×g for 10 min) and theresulting cell pellet is to be resuspended in 0.5 ml of heparinisedsaline. The total numbers of white blood cells are to be determined inBAL fluid and blood using a blood cell counter and adjusted to 1×10⁶cell/ml. Differential cell count was calculated manually. Fortymicroliters of the cell suspension is to be centrifuged using cytospin 3to prepare a cell smear. The cell smear is to be stained with a bloodstaining solution for differentiation and microscopically observed toidentify eosinophil according to their morphological characteristics.The number of each cell type among 300 white blood cells in the cellsmear has to be determined and expressed as a percentage, and the numberof neutrophils and macrophages in each BALf are to be calculated.

Assay 14: Reversal of Corticosteroid Insensitivity in Cigarette SmokeExtract Induced Pulmonary Inflammation (COPD) Model

Female Balb/c mice are to be acclimatized for seven days prior to thestart of the experiment. Animals are then to be randomly distributed tovarious groups based on their body weights. On day 1, animals are to beanaesthetised with ether and cigarette smoke extract is to beadministered by intranasal route in volume of 50 μl/mouse and animalsare to be exposed to CSE daily for next five days (d1 to d6). On day 7,mice are to be administered by dexamethasone at 10 mg/kg by oral gavageand 60 mins later, mice are to be administered with CSE by intranasalroute and it has to be repeated for next four days (d7 to d11). From day9 to day 11, animals are to be administered by test compound or vehicleby oral/intranasal route and 30 mins after dexamethasone administrationand 30 mins later animals are to be anaesthetised with ether andcigarette smoke extract is to be administered by intranasal route involume of 50 μl/mouse and animals are to be exposed to CSE daily afterthe test compound administration for next two days (i.e. d9 to d11), ond12, twenty four hours after last CSE exposure animals are to beexsanguinated under anesthesia, and the trachea is to be cannulated andthe lungs are to be lavaged with 0.5-ml aliquots of heparinised PBS (1unit/ml) four times through tracheal cannula (total volume 2 ml). BALhas to be stored at 2-8° C. until assayed for total cell anddifferential leukocyte count. Bronchioalveolar fluid is to becentrifuged (500×g for 10 min) and the resulting cell pellet has to beresuspended in 0.5 ml of heparinised saline. The total number of whiteblood cells is to be determined in BAL fluid and blood using a bloodcell counter and adjusted to 1×10⁶ cell/ml. Differential cell count isto be calculated manually. Forty microliters of the cell suspension isto be centrifuged using cytospin 3 to prepare a cell smear. The cellsmear is to be stained with a blood staining solution fordifferentiation and microscopically has to be observed to identifyeosinophil according to their morphological characteristics. The numberof each cell type among 300 white blood cells in the cell smear are tobe determined and will be expressed as a percentage, and the number ofneutrophils and macrophages in each BAL fluid are to be calculated.

Assay 15: Acute Cigarette Smoke Induced Cell Infiltration in Male Balb/cMice

Animals (male Balb/c mice) are to be acclimatized for seven days priorto the start of the experiment. Animals are then to be randomlydistributed to various groups based on their body weights. On day 1,mice is to be administered test compound or vehicle by oral/intranasalroute and after 1 hr test compound administration animals are to beplaced in whole body exposure box. On day 1 and d2 mice are exposed tothe mainstream smoke of 6 cigarettes and of 8 cigarettes on day 3, andof 10 cigarettes on day 4. Exposure to the smoke of each cigarette lastsfor 10 min (cigarette are to be completely burned in the first twominutes and followed by an air flow with animal ventilator and next 20min exposure with fresh room air. After every second cigarette anadditional break of 20 min with exposure to fresh room air is to beconducted. Control animals are to be exposed to room air chamber. Fromday 1 to d4 animals are administered by test compound either oral orintranasal route. On day 5, 24 hours after last cigarette smoke (CS)exposure animals are exsanguinated under anesthesia, and the trachea isto be cannulated and the lungs are lavaged with 0.5-ml aliquots ofheparinised PBS (1 unit/ml) four times through tracheal cannula (totalvolume 2 ml). Bronchioalveolar (BAL) collected is to be stored at 2-8°C. until assayed for total cell and differential leukocyte count. BALfluid is to be centrifuged (500×g for 10 min) and the resulting cellpellet is resuspended in 0.5 ml of heparinised saline. The total numberof white blood cells is to be determined in BAL fluid and blood using ablood cell counter and adjusted to 1×10⁶ cell/ml. Differential cellcount is calculated manually. Forty microliters of the cell suspensionis centrifuged using cytospin 3 to prepare a cell smear. The cell smearis stained with a blood staining solution for differentiation andmicroscopically observed to identify eosinophil according to theirmorphological characteristics. The number of each cell type among 300white blood cells in the cell smear are to be determined and expressedas a percentage, and the number of neutrophils and macrophages in eachBAL fluid are to be calculated.

Results:

As depicted in FIGS. 7A and 7B, Compound A1 reduced macrophages andneutrophil infiltration into BALF thereby indicating a therapeutic rolein chronic obstructive pulmonary diseases

Assay 16: Ovalbumin-Induced Nasal Eosinophil and Neutrophil Accumulationin Mice

Animals (mice) are to be acclimatized for seven days prior to the startof the experiment. Animals are then to be randomly distributed tovarious groups based on their body weights. Animals are to be immunizedwith OVA (40 μg/kg i.p.) on day 1 and 5. In order to elicit localinflammatory responses in the nose, mice are to be repeatedly challengedintra-nasally (10 μL/per nostril) on days 12-19 with OVA (3% OVA insaline). On day 19 non-fasted mice are to be dosed intra-nasally (10μL/nostril) with either vehicle or test compound 2 hours before to thestart of the final OVA challenge. Two hrs later, each animal is to bereceived a final intranasal OVA (3%) challenge). After a further 8 hr,each animal is to be anaesthetized and nasal lavage is to be carried outby instilling 1 ml of PBS into the posterior nares via a rostrallyimplanted tracheal cannula extending to a position that is approximately1 mm before the posterior nares. This procedure has to be repeated togive a yield of approximately 2 ml of lavage fluid. Total cell numbersin the nasal lavage fluid samples are to be measured using ahaemocytometer. Cytospin smears of the nasal lavage fluid samples are tobe prepared by centrifugation at 1200 rpm for 2 min at RT and stainedusing a Diff-Quik stain system (Dade Behring) for differential cellcounts. Cells are to be counted using oil immersion microscopy.

Assay 17: Poly-LC-Induced Cell Accumulation in Mice

Specific pathogen-free A/J mice (males, 5 weeks old) are to beacclimatized for seven days prior to the start of the experiment.Animals are then to be randomly distributed to various groups based ontheir body weights Animals are to be administered with poly (1:C)-LMW(poly-IC; 1 mg/mL, 40 μL) intranasally twice daily for 3 days underanaesthesia with 3% isoflurane Animals are to be treated with testcompound by intra-nasally (35 μL of solution in 50% DMSO/PBS) 2 hrbefore each poly-1:C treatment. Twenty four hr after the last poly-1:Cchallenge, animals are to be anesthetized, the trachea has to becannulated and BALF is to be collected. The concentrations of alveolarmacrophages and neutrophils in BALF are to be determined by using ablood cell counter and adjusted to 1×10⁶ cell/ml. Differential cellcount is calculated manually. Forty microliters of the cell suspensionis centrifuged using cytospin 3 to prepare a cell smear. The cell smearis stained with a blood staining solution for differentiation andmicroscopically observed to identify eosinophil according to theirmorphological characteristics. The number of each cell type among 300white blood cells in the cell smear are to be determined and expressedas a percentage, and the number of neutrophils and macrophages in eachBAL fluid are to be calculated.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as described above. It is intended that theappended claims define the scope of the invention and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

Pharmaceutical Composition of Compound A1 Example I

The capsules described below containing 5 or 10 mg of Compound A1 areprepared.

Ingredients % w/w Compound A1 4.5 Microcrystalline cellulose (AvicelPH102) 83.8 Hydroxypropyl cellulose (Klucel LF) 4.5 Purified Water q.sLow substituted hydroxylpropyl cellulose (L-HPC; LH-11) 5.9 Talc 0.3Colloidal silicon dioxide (Aerosil-200) 0.3 Magnesium stearate 0.6

Example II

The capsules described below containing 25, 50, or 100 mg of Compound A1are prepared.

Ingredients % w/w Compound A1 22.7 Microcrystalline cellulose (AvicelPH102) 67.2 Hydroxypropyl cellulose (Klucel LF) 3.4 Purified Water q.sLow substituted hydroxylpropyl cellulose (L-HPC; LH-11) 5.7 Talc 0.2Colloidal silicon dioxide (Aerosil-200) 0.2 Magnesium stearate 0.6

All publications and patent and/or patent applications cited in thisapplication are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated herein by reference.

What is claimed is:
 1. A composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof, wherein the compositionis substantially free of(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneand pharmaceutically acceptable salts thereof.
 2. A compositioncomprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one,wherein the composition is substantially free of(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one.
 3. A composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof, wherein the compositionhas an enantiomeric excess greater than about 90% relative to(R)-2-(1-(9H-purin-6-ylamino)propyI)-3-(3-fluorophenyl)-4H-chromen-4-one.4. A composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one,wherein the composition has an enantiomeric excess greater than about90% relative to(R)-2-(1-(9H-purin-6-ylamino)propyI)-3-(3-fluorophenyl)-4H-chromen-4-one%.
 5. A pharmaceutical composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable carrier, wherein the composition issubstantially free of(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneand pharmaceutically acceptable salts thereof.
 6. A pharmaceuticalcomposition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneand at least one pharmaceutically acceptable carrier, wherein thecomposition is substantially free of(R)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one.7. A pharmaceutical composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable carrier, wherein the composition has anenantiomeric excess greater than about 90% relative to(R)-2-(1-(9H-purin-6-ylamino)propyI)-3-(3-fluorophenyl)-4H-chromen-4-one.8. A pharmaceutical composition comprising(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneand at least one pharmaceutically acceptable carrier, wherein thecomposition has an enantiomeric excess greater than about 90% relativeto(R)-2-(1-(9H-purin-6-ylamino)propyI)-3-(3-fluorophenyl)-4H-chromen-4-one.9. The composition of claim 3, wherein the composition has anenantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 95%.10. The composition of claim 3, wherein the composition has anenantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 98%.11. The composition of claim 3, wherein the composition has anenantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 99%.12. The composition of claim 4, wherein the composition has anenantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 95%.
 13. The composition of claim 4, wherein thecomposition has an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 98%.
 14. The composition of claim 4, wherein thecomposition has an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 99%.
 15. The pharmaceutical composition of claim 7,wherein the composition has an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 95%.16. The pharmaceutical composition of claim 7, wherein the compositionhas an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 98%.17. The pharmaceutical composition of claim 7, wherein the compositionhas an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-oneor a pharmaceutically acceptable salt thereof greater than about 99%.18. The pharmaceutical composition of claim 8, wherein the compositionhas an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 95%.
 19. The pharmaceutical composition of claim 8,wherein the composition has an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 98%.
 20. The pharmaceutical composition of claim 8,wherein the composition has an enantiomeric excess of(S)-2-(1-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-onegreater than about 99%.
 21. A method of inhibiting a catalytic activityof a PI3 δ kinase present in a cell, comprising contacting the cell withan effective amount of a composition of claim
 1. 22. A method ofinhibiting a catalytic activity of a PI3 γ kinase present in a cell,comprising contacting the cell with an effective amount of a compositionof claim
 1. 23. A method of inhibiting a catalytic activity of a PI3 δkinase and PI3 γ kinase present in a cell, comprising contacting thecell with an effective amount of a composition of claim
 1. 24. Themethod of claim 21, wherein the inhibition takes place in a cell,wherein the cell is in a subject suffering from leukemia.
 25. A methodof ameliorating leukemia, the method comprising administering aneffective amount of a composition of claim 1 to a patient in needthereof.
 26. The method of claim 25, wherein the leukemia is acutelymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia (CLL), or acute myeloid leukemia (AML).
 27. A method ofinhibiting a catalytic activity of a PI3 δ kinase present in a cell,comprising contacting the cell with an effective amount of apharmaceutical composition of claim
 5. 28. A method of inhibiting acatalytic activity of a PI3 γ kinase present in a cell, comprisingcontacting the cell with an effective amount of a pharmaceuticalcomposition of claim
 5. 29. A method of inhibiting a catalytic activityof a PI3 δ kinase and PI3 γ kinase present in a cell, comprisingcontacting the cell with an effective amount of a pharmaceuticalcomposition of claim
 5. 30. The method of claim 27, wherein theinhibition takes place in a cell, wherein the cell is in a subjectsuffering from leukemia.
 31. A method of ameliorating leukemia, themethod comprising administering an effective amount of a pharmaceuticalcomposition of claim 5 to a patient in need thereof.
 32. The method ofclaim 31, wherein the leukemia is acute lymphocytic leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia (CLL), or acutemyeloid leukemia (AML).